Methods and materials for the generation of regulatory t cells

ABSTRACT

Methods are disclosed for the generation of immunosuppressive regulatory T cells. The methods can include contacting a population of CD4+CD25− T cells with a T cell receptor (TCR)/CD3 activator, a TCR co-stimulator activator, and rapamycin. Kits for the generation of immunosuppressive regulatory T cells, methods of use, and cell populations are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 13/689,530 filed Nov. 29, 2012, which is acontinuation of U.S. application Ser. No. 12/604,263, filed Oct. 22,2009, now abandoned, which is a continuation application of U.S.application Ser. No. 12/279,713, filed Oct. 30, 2009, now abandoned,which is a 371 U.S. national application of international applicationNo. PCT/IB2007/050508, filed Feb. 15, 2007, which claims priority toG.B. patent application Ser. No. 603081.1, filed Feb. 15, 2006, each ofwhich are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods and materials for the generation ofregulatory T cells.

SUMMARY OF THE INVENTION

The proliferation of T cells can be stimulated by contacting the cellswith a T cell receptor/CD3 activator, a TCR co-stimulatory activator,and rapamycin. Methods for generating regulatory T cells fromCD4+CD25+FOXP3− T cells and the use of these methods in the generationof T cell populations are disclosed. These cells can be used in avariety of applications including immunotherapy.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the extent of proliferation of various ratios of T cellscultured with or without rapamycin in a standard proliferation assay.CD25− T cells (T cells cultured without Rapamycin) were analyzed forproliferative capacity in a standard proliferation assay using ³HThymidine incorporation after 4 days stimulation. CD25+ T cells (T cellscultured with Rapamycin) were added to the CD25− T cells in a ratio from1:1 to 1:32. The proliferative capacity of CD25− T cells alone wasdefined as 100% proliferation. The x-axis is the ratio of CD25+ cells toCD25− cells. The y-axis is percent proliferation.

FIGS. 2A and 2B show flow cytometry analysis of CD4 and CD25 expressionon CD4+ T cells expanded (FIG. 2A) with rapamycin or (FIG. 2B) withoutrapamycin.

DETAILED DESCRIPTION OF THE INVENTION

While compositions and methods are described in terms of “comprising”various components or steps (interpreted as meaning “including, but notlimited to”), the compositions and methods can also “consist essentiallyof” or “consist of” the various components and steps, such terminologyshould be interpreted as defining essentially closed-member groups.

Aspects of the present invention relate to methods for generatingCD4+CD25+FOXP3− regulatory T cells and the use of these methods in thegeneration of T cell populations which have applications in for exampleimmunotherapy.

Naturally occurring regulatory T (Treg) cells suppress immune responsesand play an important role in immunotherapy against autoimmune diseasesand provide transplantation tolerance. Various populations of Treg cellshave been described and include naturally occurring CD4+CD25+FOXP3+cells and induced Tr1 and Th3 cells that secrete IL-10 and TGFβrespectively. The natural occurring CD4+CD25+FOXP3+ Treg cellsrepresents 5-10% of the CD4+ T cells in the peripheral blood and are ina hypoproliferative state which has hampered detailed characterizationand the potential use of these cells in immune therapy. In vivo usestherefore have relied on expansion protocols to generate sufficientnumbers of Treg cells for in vivo use. The clinical use of Treg cells islimited by the lack of appropriate isolation and expansions protocols togenerate sufficient numbers for in vivo infusion.

The present invention addresses this need by providing a method ofgenerating a population of immunosuppressive Treg cells from theabundant CD4+CD25− T cell population. This provides Treg cells insufficient numbers for in vivo infusions. The protocol can be used bothfor generating Treg cells for research purposes and for clinical use byinfusion in patients.

The invention thus provides a method in which Treg cells expressing CD4,CD25 but not FOXP3 are generated. In one embodiment, the protocol uses asolid support carrying CD3/CD28 antibodies, Rapamycin and optionallycytokines, such as IL-4 and/or IL-2 to activate and expand CD4+ T cellsisolated either from peripheral blood or leukopheresis products.

The use of anti-CD3/CD28, for example, provides the activation signalfor the T cell population. T cells require at least two signals foractivation. Signal one is antigen specific and is elicited bypeptide/major histocompatibility complex (MHC) complexes presented byantigen-presenting cells (APC) and received through the T-cell receptor(TCR)/CD3 complex. Signal two (which is antigen non-specific) is alsodelivered by antigen presenting cells and one of the candidate moleculesfor its receptor is the T cell antigen CD28. It is thought that whenboth the TCR/CD3 and CD28 T cell receptors are occupied by appropriateligands, T cells are stimulated to proliferate and produce IL-2 (acytokine essential for T cell proliferation), whereas occupation of theT cell receptor alone favors T cell anergy or apoptosis.

In vitro it has been shown that T cell growth and cytokine productioncan be stimulated by culturing T cells with anti-CD3 antibodies whichhave been immobilized to a solid phase (for example beads or tissueculture plates) and adding soluble CD28 antibodies (Sommer et al., 1993.Eur. J. Immunol. 23:2498-2502, Sunder-Plassmann et al., 1996. Blood87:5179-5184). More recently it has been shown that co-immobilising bothCD3 and CD28 antibodies to the same solid phase or to different solidphases can also induce T cell proliferation (Levine et al., 1997. J. ofImmunol. 159: 5921-30; Li et al., 1999. Science 283:848-851).

Rapamycin is an immunosuppressive agent used to prevent allograftrejection. Recently, the cellular target for Rapamycin in vitro has beendiscovered, and shown to selectively expand naturally occurringCD4+CD25+FOXP3+ regulatory T cells. The present inventors have developeda new protocol using Rapamycin to generate new Treg cells from CD4+CD25−T cells. In contrast to naturally occurring CD4+CD25+ Treg cells thatexpress FOXP3, the Treg cells generated in accordance with the inventiondo not express FOXP3 but still show very strong suppressive capacities.

Treg cells generated according to the invention have many potentialuses, including experimental and therapeutic uses. In particular it isenvisaged that such T cell populations will be extremely useful insuppressing undesirable or inappropriate immune responses. In suchmethods a small number of T cells are removed from a patient and thenmanipulated and expanded ex vivo before reinfusing them into thepatient. Examples of diseases which may be treated in this way areautoimmune diseases and conditions in which suppressed immune activityis desirable, e.g. for allo-transplantation tolerance. A therapeuticmethod could comprise providing a mammal, obtaining CD4+CD25− T cellsfrom the mammal; expanding/activating the T cells ex vivo in accordancewith the methods of the invention as described above; and administeringthe expanded/activated T cells to the mammal to be treated. The firstmammal and the mammal to be treated can be the same or different. Themammal can generally be any mammal, such as cats, dogs, rabbits, horses,pigs, cows, goats, sheep, monkeys, or humans. The first mammal (“donor”)can be syngeneic, allogeneic, or xenogeneic. Therapy could beadministered to mammals having aberrant immune response (such asautoimmune diseases including, for example diabetes, multiple sclerosis,myasthenia gravia, neuritis, lupus, rheumatoid arthritis, psoriasis, andinflammatory bowel disease), tissue transplantation, or fertilitytreatments.

The main technical hurdles involved in such therapies include thepurification of the cells of interest from the patient and the expansionand/or the manipulation of the cells in vitro. Such therapies generallyrequire a large number of cells and thus it can be seen that it is vitalto optimize the methods of inducing in vitro T cell proliferation inorder to maximize the number of T cells produced and minimize the timerequired to produce the T cells in sufficient numbers.

Thus viewed in a first aspect the present invention provides a methodfor stimulating proliferation of CD4+CD25− T cells comprising contactingthe cells with a T cell receptor (TCR)/CD3 activator, a TCRco-stimulator activator and rapamycin. The cells can be cultured underconditions and for a time suitable to achieve levels of proliferation asdescribed hereinafter. This method generates a CD4+CD25+ T cellpopulation. T cells which are FOXP3− may be used and the generated Tcell population in that case remain FOXP3−.

An alternative embodiment of the invention provides a method ofpreparing a T cell population of CD4+CD25+ T cells, preferablyCD4+CD25+FOXP3− T cells, comprising contacting CD4+CD25− T cells(preferably CD4+CD25−FOXP3− cells) with a TCR/CD3 activator, a TCRco-stimulatory activator and rapamycin to stimulate proliferation of theCD4+CD25− T cells and optionally isolating the T cell population. TheCD4+CD25+ (preferably CD4+CD25+FOXP3−) T cell population thus formedprovides a further aspect of the invention. Preferably, the T cellpopulation thus formed is immunosuppressive. Thus, the cells are able tosuppress proliferation of syngeneic T cells in vitro, e.g. as describedin the Examples herein. At a 1:1 ratio of the test T cells, with, forexample, T cells prior to stimulation, immunosuppressive cellspreferably achieve at least 70, 80 or 90% suppression of proliferation,i.e. reduction in cell numbers relative to control over a suitableculture period, e.g. between 2 and 14 days, for example 4 days.Preferably, the CD4+CD25− T cells used in the stimulation methods andthe CD4+CD25+ T cells generated according to the invention as describedherein are FOXP3−.

The TCR/CD3 activator can be an antibody or ligand for TCR/CD3, forexample a CD3 antibody. The TCR co-stimulatory activator can be anantibody or ligand for CD28, CD137 (4-1BB), GITR, B7-1/2, CD5, ICOS,OX40 or CD40. Preferably the TCR/CD3 activator is a CD3 antibody and theTCR co-stimulatory activator is a CD28 antibody.

Preferably the activators, e.g. antibodies, are immobilized to a solidphase. The activators, e.g. antibodies may be provided at variableconcentration on the solid support, such as at a ratio of about 1:10 toabout 10:1 of CD28 antibody to TCR/CD3 antibody. Optionally more thanone of the TCR/CD3 activators and/or more than one of the TCRco-stimulatory activators may be used in methods of the invention.

“Stimulating proliferation” as used herein refers to any event whichresults in a detectable increase or expansion in the number of T cellspresent when compared with the number present in the absence of suchstimulation. The daughter cells which are generated by the proliferationmay have a different phenotype as a result of stimulation, inparticular, stimulation results in CD25 expression thus resulting in thegeneration of CD4+CD25+ cells from CD4+CD25− cells.

Such an increase in number may be relatively small, but is preferably asignificant increase such as for example an increase in cell number ofat least about 2 fold, preferably at least about 5 fold, about 20 foldor about 50 fold and more preferably at least about 100 fold, about 500fold, or greater than about 1000 fold. Such increases in number may bemeasured at any appropriate time point in the cell expansion protocol,such as for example at day 4 up to day 12 of the cell expansionprotocol.

T cells which are considered positive for a specific antigen, e.g. CD3,carry detectable levels of the antigen, e.g. as determined by cellsorting (e.g. flow cytometry) or the use of a solid support to which anappropriate binding partner, e.g. antibody is bound.

As discussed above stimulation of T cells with a TCR/CD3 activator and aTCR co-stimulatory activator, e.g. anti-CD3 and anti-CD28 antibodiesalso provides the two signals required for T cell activation. Thus,viewed in a yet further alternative way, the present invention providesa method of activating CD4+CD25− T cells comprising contacting the cellswith a TCR/CD3 activator and a TCR co-stimulatory activator (e.g. CD28antibodies and T cell receptor (TCR)/CD3 antibodies) and rapamycin.

Thus, throughout the discussion of the present invention, reference tomethods of stimulating proliferation and use of T cells so stimulatedshould also be read to include methods of stimulating T cell activationand the use of such activated T cells.

Any CD4+CD25− T cell population may be expanded/ activated using thepresent method. For example the T cell population may compriseCD4+CD25-CD8+ T cells. In a preferred aspect, CD4+CD25− T cells areseparated from the source starting material prior to stimulation andexpansion. Thus, in a preferred aspect the present invention provides amethod for stimulating proliferation of CD4+CD25− T cells comprising atleast the steps of: (i) isolating CD4+CD25− T cells from a sample, and(ii) contacting the cells with a TCR/CD3 activator and a TCRco-stimulatory activator (e.g. CD28 antibodies and T cell receptor(TCR)/CD3 antibodies) and rapamycin.

An additional embodiment of the invention provides a method of preparinga T cell population of CD4+CD25+ (preferably CD4+CD25+FOXP3−) T cells,comprising (i) isolating CD4+CD25− T cells from a sample, and (ii)contacting the cells with a TCR/CD3 activator and a TCR costimulatoryactivator (e.g. CD28 antibodies and T cell receptor (TCR)/CD3antibodies) and rapamycin to stimulate proliferation of the CD4+CD25− Tcells and optionally isolating the T cell population.

The CD4+CD25− cells which are used in methods of the invention maycomprise the entire CD4+CD25− T cell population or a portion of thatpopulation. For example, a sub-population may be used in the method,e.g. CD4+CD25−FOXP3−, which sub-population may be used in its entiretyor a portion of that sub-population may be used. Thus the isolatedCD4+CD25− cells subjected to the stimulation method of the invention maycontain at least about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, or about 90% of the CD4+CD25− cells (or asub-population thereof) in the sample from which the CD4+CD25− cellswere isolated. Preferably however, in order to generate sufficientlevels of Treg for in vivo uses, essentially the entire CD4+CD25−population from the starting sample is used in methods of the invention.

The stimulation method may be performed in the presence of other cells,e.g. other T cells, such as CD4+CD25+ cells which themselves willproliferate during the method of the invention. Thus in the abovedescribed protocol, the step of isolating CD4+CD25− T cells may compriseisolating a significant portion (i.e. at least 20, 30, 40, 50, 60, 70,80 or 90%) or all CD4+ cells, i.e. which include both CD25− and CD25+,from the sample. Thus in a preferred embodiment of the invention, theisolation step comprises the isolation of CD4+ cells. Furthermore, othercells may also be present such that the e.g. CD4+, CD4+CD25− and/orCD4+CD25−FOXP3− cells form only a portion of the cells used in thestimulatory method, before or after the isolation step. Thus in thestimulatory step the CD4+CD25− may be present as a portion of the cellssubject to stimulation, i.e. an enriched preparation may be used, suchas comprising at least about 50, about 60, about 70, about 80 or about90% of the total cells subjected to stimulation.

Preferably however, at least some CD4+CD25+ cells are absent, e.g. atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, or about 90% of the cells which appeared in thestarting material are absent. Alternatively expressed the cells forstimulation/expansion are preferably substantially all CD4+CD25− cells,such as at least about 80%, about 90%, about 95% or about 98% CD4+CD25−T cells are used in the method. Especially preferably, at least someCD4+FOXP3+ cells are absent in the cells used for expansion, e.g. atleast 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the cells which appearedin the starting material of the sample are absent. Alternativelyexpressed the cells for stimulation/expansion are preferablysubstantially all CD4+FOXP3− (preferably CD4+CD25−FOXP3−) T cells, e.g.at least 80, 90, 95 or 98% CD4+FOXP3− or CD4+CD25-FOXP3− T cells areused in the method.

Sources of T cells and methods of isolating particular T cellpopulations (e.g. CD4+ cells) which can be expanded by stimulationaccording to the methods of the present invention are well known anddescribed in the literature. Thus for example T cells may convenientlybe isolated from the blood e.g. from a peripheral blood mononuclear cell(PBMC) population isolated from blood, or from other blood-derivedpreparations such as leukopheresis products or from bone marrow, lymph,thymus, spleen or umbilical cord. Examples of T cell populations whichcan be selected and expanded according to the methods of the presentinvention include those which are derived by negative selection fromPBMC where B cells and monocytes are depleted, (optionally negativeselection of CD25− and/or FOXP3− cells), positive selection of the CD4+T cells using beads coupled with Abs against CD4 (or flow cytometry) andpositive selection of T cells using sheep red blood cells. T cellpopulations may be derived from any appropriate source, including humanor animal sources.

Immobilization of the activators (e.g. CD28 and CD3/TCR antibodies) to asolid phase according to the present invention means that the activatorsare immobilized to (or on) the same or different solid supports.Preferably, the antibodies are co-immobilized to (or on) the samesurface.

“Immobilized” or “Immobilization” as used herein refers to any means bywhich activators, e.g. antibodies can be bound, attached or otherwisefixed to a solid phase. Such immobilization may be direct (i.e. theactivators themselves are attached to the solid phase) or indirect (i.e.the activators are attached via an intermediate entity) and may occur byway of any chemical or non-chemical attachment method. Such methodsinclude immobilization of activators by adsorption and/or by covalentattachment or via affinity between molecules (e.g. immobilization on anavidin-coated surface of biotinylated antibodies, or the immobilizationof an anti CD3 or anti CD28 via a secondary antibody or antibodies whichare themselves attached to the solid phase). The immobilization must beof sufficient strength that the activators are not removed under theconditions used to prepare the solid phase with the activators attached(for example the immobilized activators must withstand the washingconditions etc. associated with the preparation of the solid phase) andare also not removed under the conditions used to stimulate T cellproliferation.

The step of “contacting” the population of T cells with the activators,e.g. immobilized to the solid phase, may be carried out in anyconvenient or desired way. When the method is performed withoutimmobilized activators, the activators are added into the solutioncontaining the cells to be expanded. When one or more of the activatorsis carried on a solid support, e.g. if the solid phase was a tissueculture plate or flask or some other flat surface, the population of Tcells, conveniently in an aqueous medium such as for example anappropriate cell culture medium, may simply be added to the solid phaseunder appropriate conditions. Alternatively, if the solid phase isparticulate (for example beads) the solid phase itself may be added tothe T cell population under appropriate conditions. In any event, in thecontacting step, the cells are contacted with activators which arealready immobilized i.e. the activators are bound to the solid supportbefore they bind to the cells, i.e. before the contacting step.

The activators are provided in a molar equivalent or molar excess to thecells to be stimulated. Thus, for example in the case ofanti-CD3/anti-CD28 solid supports, e.g. beads, the bead to T cell ratiomay range from 1:10 to 1:1, e.g. 3:1. Beads for this purposes are wellknown in the art (e.g. from Dynal Biotech ASA).

Appropriate conditions will be such conditions which are suitable for Tcell growth and should be chosen depending on for example the T cellpopulation concerned and the particular cell culture medium used. Suchmedia and conditions are well known in the art. Typical conditions maybe maintenance of cells in a humidified atmosphere containing 5 or 10%CO₂ at 37° C. or under low O₂ concentration (e.g. 2%). Appropriateculture medium is selected depending on the T cell population beingexpanded. For example for T cell populations the culture medium×vivo 15supplemented with 5% human AB serum is appropriate or RPMI 1640 withFCS, CellGro DC, or or other media suited for cell culture may be used.

Rapamycin is contacted with the cells prior to, simultaneously with,and/or subsequent to contact of the cells with the activators. Rapamycinis preferably present throughout the proliferation/expansion step in themethod according to the invention. The rapamycin may be added in one ormore steps. Thus for example, as described in the examples herein,isolated CD4+ cells may be stimulated with activators (CD3 antibodiesand CD28 antibodies) and rapamycin at the same time. In this method,subsequent growth and passaging is performed in the presence ofrapamycin, but not the activators.

Rapamycin may be used at a concentration of from about 0.01 μM to about10 μM, such as about 0.5 μM to about 2 μM, or about 1 μM. Rapamycin is aprotein kinase inhibitor with a molecular weight of 914.2, also referredto as Sirolimus, Rapamune, AY-22989, RAPA and NSC-226080, available fromSigma, Calbio Chem, LC Labs etc. Rapamycin is available from a varietyof commercial sources, such as A.G. Scientific, Inc. (San Diego, Calif.,USA).

Other cytokines and/or growth factors may be added to the cultures asappropriate. Such cytokines and/or growth factors are added atappropriate concentrations and time points. For example IL-2 and/or IL-4may be added to enhance the proliferation of the T cells and othercytokines may be added to induce particular differentiation patterns ifrequired (e.g. TGF-(and IL10). For example IL-4 has been shown totrigger differentiation of T cell populations into the Th2 subpopulationand IFN-□ to trigger differentiation into the Th1 subpopulation(Sunder-Plassmann, supra). Thus in preferred embodiments of theinvention, a cytokine, e.g. IL-2 is preferably added, e.g. at a finalconcentration of 10-2,000 U/ml, e.g. at 20 U/ml during stimulation andat 1,000 U/ml periodically during the initial culture period and 20 U/mlin the period prior to harvest. IL-4 is preferably added, e.g. at afinal concentration of 1,000-5,000 U/ml, e.g. at 1,000 U/ml duringstimulation periodically during the initial culture period and in theperiod prior to harvest. Appropriate cytokines and growth factors andtheir effects on T cells are well known and described in the art. Oncethe T cell population has been brought into contact with the activatorsand rapamycin under appropriate conditions for growth of the T cells,growth is allowed to progress for a time period selected according tothe final number of T cells required and the rate of expansion of thecells. Passaging of the cells may be undertaken during this period. Sucha time period is normally between 3 and 10 days but can be as long as 14to 20 days or even longer providing the viability and continuedproliferation of the T cells is maintained.

If necessary the T cell population which is being expanded/activatedaccording to the methods of the present invention may be re-stimulatedby contacting the cells with further activators in a similar way to theinitial stimulation. In general, re-stimulation is only necessary ordesired if the T cells are to be cultured for a long period of time,e.g. more than 20 days.

Treg cells having immunosuppressive properties can be assessed byanalysis of their phenotype (CD4+CD25+FOXP3−) and their ability tosuppress proliferation of syngeneic T cells in vitro, e.g. as describedin the Examples herein.

Once a T cell population has been expanded to a required level asdescribed above, the expanded population can then be separated from thesolid phase in an appropriate way. For example, if the activators areattached to a solid phase and that solid phase is a tissue culture well,plate or bottle, the T cell population can be obtained by removal of theculture medium which contains the T cells. If the solid phase comprisesfor example magnetic beads, a magnetic field is used to attract thebeads to the side of the vessel and the culture medium containing the Tcells can then be poured off. Other particulate solid supports e.g.non-magnetic beads may be centrifuged or filtered away from the cells.Although the majority of T cells will be located in the culture medium,some T cells are likely to be attached to the solid phase afterexpansion. If desired such T cells can be detached by for exampleresuspension using a pipette or other suitable means. Such aresuspension will normally be carried out before the T cells areseparated from the solid phase to improve the yield. Once separated fromthe solid phase the T cell population can then be further treated and/ormanipulated in any desired way or used directly for suitableapplications such as for example in vitro experiments and research,non-therapeutic applications, therapeutic applications etc. as discussedbelow.

When soluble activators are employed, these may be removed bycompetition with appropriate ligands, e.g. CD3 or CD28, but morepreferably the T cells are collected from the culture medium and usedfor applications as described herein without further refinement.Conveniently, for large scale applications, appropriate isolation andpreparation platforms may be used for selection of the T cellpopulations for stimulation and/or for the expansion protocol and/orisolation of the generated T cell population. In this regard specialmention may be made of Dynal's ClinExVivo™ platforms in which closedsterile disposable bags may be used for any magnetic cell separationsteps which are performed, e.g. for cell isolation prior to, or after,expansion.

Thus in a preferred aspect the present invention provides a method forstimulating proliferation of CD4+CD25− T cells comprising at least thesteps of: (i) isolating CD4+CD25− T cells from a sample, preferably by amethod comprising at least the steps of contacting the sample with asolid support carrying CD4 antibodies and isolating the T cells whichbind to the solid support, or using negative isolation of CD4+ T cellsby removing all unwanted cells with a mix of mAbs followed by magneticbeads to capture cells (ii) contacting the cells with a) a solid supportcarrying CD28 antibodies and TCR/CD3 antibodies and b) rapamycin, (iii)incubating the cells under conditions to allow proliferation, preferablyin the presence of IL-4 and/or IL-2 and (iv) isolating the T cells afterthe incubation.

When the activator used in accordance with the invention is an antibodyreactive with TCR co-stimulatory activators: CD28, CD137 (4-1 BB), GITR,B7-1/2,C D5, ICOS, OX40, CD40 or CD137 or TCR/CD3 activators: CD3 or TCRmay be used. More than one antibody from each group may be used.Preferably a TCR or CD3 antibody is used in conjunction with a CD28antibody. Thus, using CD28 and TCR/CD3 antibodies as an example, a “CD28antibody” and a “TCR/CD3 antibody” according to the present inventionare antibodies capable of binding to (or immunoreactive with) CD28 orTCR/CD3 respectively. Preferred are antibodies capable of bindingspecifically to CD28 or TCR/CD3 in a manner which distinguishes from thebinding to other “non-target” molecules, i.e. preferred antibodies arethose which exhibit detectable binding affinity for CD28 or CD3/TCR butwhose binding to other molecules, for example other cell surfacemolecules is negligible, insignificant or non-detectable. In preferredaspects of the invention, the method of the invention may involve theuse of antibodies to (i) CD3 and CD28, (ii) CD3 and CD137, or (iii) CD3and CD28 and CD137.

A number of CD28, CD137 (4-1BB), GITR, B7-1/2, CD5, ICOS, OX40, CD40,CD3 or TCR antibodies (or anti-CD28 etc.) are known in the literature orcommercially available. Any such antibody, or its fragments orderivatives may be used (so long as they retain binding activity).

Antibodies for use in methods of the present invention may be of anyspecies, class or subtype providing that such antibodies can react withthe target of interest, e.g. CD3, the TCR, or CD28 as appropriate.

Thus “antibodies” for use in the present invention include:

(a) any of the various classes or sub-classes of immunoglobulin, e.g.IgG, IgA, IgM, IgD or IgE derived from any animal e.g. any of theanimals conventionally used, e.g. sheep, rabbits, goats, or mice, or eggyolk

(b) monoclonal or polyclonal antibodies

(c) intact antibodies or fragments of antibodies, monoclonal orpolyclonal, the fragments being those which contain the binding regionof the antibody, e.g. fragments devoid of the Fc portion (e.g. Fab,Fab′, F(ab′)2, scFv), the so called “half molecule” fragments obtainedby reductive cleavage of the disulphide bonds connecting the heavy chaincomponents in the intact antibody. Fv may be defined as a fragmentcontaining the variable region of the light chain and the variableregion of the heavy chain expressed as two chains.

(d) antibodies produced or modified by recombinant DNA or othersynthetic techniques, including monoclonal antibodies, fragments ofantibodies, “humanised antibodies”, chimeric antibodies, orsynthetically made or altered antibody-like structures. Also includedare functional derivatives or “equivalents” of antibodies e.g. singlechain antibodies, CDR-grafted antibodies etc. A single chain antibody(SCA) may be defined as a genetically engineered molecule containing thevariable region of the light chain, the variable region of the heavychain, linked by a suitable polypeptide linker as a fused single chainmolecule.

Methods of preparation of antibody fragments and synthetic andderivatized antibodies are well known in the art and widely described inthe literature and will not be described herein.

In a preferred embodiment of the invention CD3 and CD28 antibodies areused and more preferably these antibodies are monoclonal antibodies.Preferred CD28 antibodies are L293 (Becton Dickinson), Mab 9.3 (an IgG2Aantibody, Dr. Ledbetter, Bristol Myers Squibb Corporation, Seattle,Wash.), Mab Kolt-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM), YTH913.12(Monosan), CD28.2 (Coulter), EX5.3D10 (IgG2A), and B-T3 clone (Diaclone,France). Preferred CD3 antibodies are Spv.T3b, OKT-3 (OrthoPharmaceutical), MEM-57 and WT32 (Monosan), SK7 (Becton Dickinson),UCHTI (Coulter), HIT3a (Pharmigan), and clone BC3 (Fred HutchinsonCancer Research Centre, Seattle). Preferred TCR antibodies are the cloneBL-A (Monosan).

Solid phases for use in the present invention may be any solid surfacesto which activators, e.g. antibodies or antibody fragments can beimmobilized either directly (i.e. the antibodies themselves are attachedto the solid phase) or indirectly (i.e. the antibodies are attached viaan intermediate entity, for example a secondary antibody, which isitself attached to the solid phase). For example, such solid phases maycomprise glass, silica, latex, polymeric materials, plastic, tissueculture plastic, dextran, cellulose and PEG, iron and other metals. Suchsolid supports may take the form of any of the well known supports ormatrices which are currently used for immobilization or separation, forexample particles, beads, bottles, tubes, strips plates or wells,sheets, fibres, capillaries, needles, combs, pipette tips, microarrays,chips, filters, membranes, and so on.

Preferably the solid phase is a particulate material. Conveniently, aparticulate solid support used according to the invention will comprisespherical beads. The size of the beads or indeed any other particulateform of support is not critical, but they may for example be of theorder of diameter of at least 1 and preferably at least 2 μm, and have amaximum diameter of preferably not more than 10 μm and more preferablynot more than 6 μm. For example, preferred beads for use in the presentmethods have a diameter of 4.5 μm though smaller particles such as thosewith a diameter of 1 μm or 2.8 μm may be used. Small particles, forexample beads of diameter less than 1 μm, may be cross-linked to providelarger complexes.

Monodisperse particles, that is those which are substantially uniform insize (e.g. size having a diameter standard deviation of less than 5%)have the advantage that they provide very uniform reproducibility ofreaction. Monodisperse polymer particles produced by the techniquedescribed in U.S. Pat. No. 4,336,173 are especially suitable.

Non-magnetic polymer beads suitable for use in the method of theinvention are available from Dynal Biotech ASA (Oslo, Norway) as well asfrom Qiagen, Amersham Pharmacia Biotech, Serotec, Seradyne, Merck,Nippon Paint, Chemagen, Promega, Prolabo, Polysciences, Agowa and BangsLaboratories.

However, to aid manipulation and separation, magnetic or magnetizablebeads are preferred. The term “magnetic” as used herein means that thesupport is capable of having a magnetic moment imparted to it whenplaced in a magnetic field, and thus is displaceable under the action ofthat field. In other words, a support comprising magnetic particles mayreadily be removed by magnetic aggregation, which provides a quick,simple and efficient way of separating the particles, and is a far lessrigorous method than traditional techniques such as centrifugation whichgenerate shear forces which may disrupt cells.

Thus, the magnetic particles may be removed onto a suitable surface byapplication of a magnetic field e.g. using a permanent magnet. It isusually sufficient to apply a magnet to the side of the vesselcontaining the sample mixture to aggregate the particles to the wall ofthe vessel.

Especially preferred particles are superparamagnetic particles asmagnetic aggregation and clumping of the particles during reaction canbe avoided. Such particles are described, for example, by

Sintef in EP-A-106873. The well-known magnetic particles sold by DynalBiotech ASA (Oslo, Norway) under the trade mark DYNABEADS®, areparticularly suited for use in the present invention. Particularlypreferred beads for use in the present invention are Dynabeads M-450.

Functionalized coated particles for use in the present invention may beprepared by modification of the beads according to U.S. Pat. Nos.4,336,173, 4,459,378 and 4,654,267 or by other procedures known in theart. Thus, beads, or other supports, may be prepared having differenttypes of functionalized surface, for example positively or negativelycharged, hydrophilic or hydrophobic. Hydrophobic surfaces (for examplehydrophobic resins) are particularly preferred for use in the attachmentof antibodies, as, by way of a hydrophobic interaction, the antibodieswill be adsorbed to the surface of the beads. This adsorption isoptionally followed by chemical linkage, for example covalent linkage ofthe antibodies to the surface of the beads. Methods for forming suchchemical or other linkages are well known and documented in the art.

Based on the varying physical and/or chemical properties of theactivators, e.g. antibodies used, such as hydrophobicity and isoelectricpoint, binding profiles will vary slightly for different activators.This is dealt with by adapting the reaction conditions in order toobtain the required levels of activators, e.g. by modifying the ratio ofactivators immobilized on the solid surface, when a solid support isused. An indication of the ratios of activators which become bound to asolid phase under certain reaction conditions can be assessed by aperson skilled in the art for example by immobilizing anti-CD3 andanti-CD28 antibodies of different sub-classes onto the solid phase undercertain conditions and then using individual reagents which reactspecifically with antibodies of the particular sub-classes in questionto determine in turn the amount and hence the ratio of the differentantibodies bound.

Magnetic or magnetizable beads are particularly preferred because of theease of manipulation. In addition, as described above, preferably thebeads have a hydrophobic surface. Thus, to prepare beads for use in themethods of the present invention the magnetic and hydrophobic beads areincubated with an appropriate mixture of the activators, e.g. CD3/(orTCR) and CD28 antibodies under appropriate reaction conditions tofacilitate adsorption and optionally chemical linkage of activators tothe surface of the beads.

The activators, in appropriate ratios, are generally mixed togetherbefore they are put into contact with the beads. “Appropriate reactionconditions” as discussed above will vary according to the particularactivators used but exemplary conditions may comprise for example anincubation of the beads and activators, e.g. antibodies in a phosphatebuffer (for example 0.5 M phosphate) of pH 7.4 and a particleconcentration of 4×10⁸ beads/ml. Human serum albumin or other serumalbumin such as bovine serum albumin may optionally be present (e.g. ata concentration of about 0.05% w/v) to stabilize the activators andblock any remaining hydrophobic patches on the surface of the beads.

Once the appropriate reaction mixture has been set up the reaction isallowed to progress for an appropriate time and under appropriateconditions to facilitate the activator absorption to the surface in therequired ratio. Again the particular conditions can be varied dependingon the components of the reaction mixture but exemplary conditionsinclude incubation for 16-24 hours at 37° C. with slow tilt androtation.

Whatever the conditions chosen, once the immobilization reaction iscomplete the beads with activators immobilized to their surfaces in theappropriate ratio can be removed from the remaining aqueous medium byplacing a magnet at the side of the reaction vessel and discarding thesupernatant. The beads will generally be washed to remove any excess nonbound antibodies and are then ready for use. Such beads, once prepared,can be used immediately or can be stored for future use.

A yet further aspect of the invention provides the use of the methods ofthe invention in the expansion/activation of T cell populations for usein in vitro experiments and research. A yet further aspect of theinvention provides the use of the methods of the invention in theexpansion/activation of T cell populations for the generation of T cellpopulations for immune suppression therapy. In such a therapy a T cellsample is taken from a patient and manipulated ex vivo as describedhereinbefore to isolate and expand an appropriate T cell populationbefore reinfusing the T cell population to a patient to suppress thepatient's immune response.

Thus, a yet further aspect of the invention provides a method oftreatment of a mammal with a condition or disease typified by anaberrant immune response or in which immune suppression would beadvantageous, comprising administering to the mammal T cells prepared inaccordance with the method of the invention. Thus in a particularembodiment, the method comprises:

a) obtaining CD4+CD25− T cells from a first mammal;

b) expanding/activating the T cells ex vivo in accordance with themethods of the invention as described above; and

c) administering the expanded/activated T cells to the mammal to betreated.

This method thus provides a method of achieving an immunosuppressiveeffect in a mammal, i.e. a method of preventing an immune response. Aneffective amount of the T cells is administered to achieve the desiredtherapeutic effect, e.g. by intravenous injection in a pharmaceuticallyacceptable diluent.

The mammal to be treated may be the same as the first mammal from whichthe T cells are sourced or may be a different mammal, i.e. a recipientof T cells from a donor. When a donor is used, the donor is preferablysyngeneic, but may also be allogeneic or even xenogeneic provided thecells are subject compatible.

The condition or disease typified by an aberrant immune response may bean autoimmune disease, for example diabetes, multiple sclerosis,myasthenia gravia, neuritis, lupus, rheumatoid arthritis, psoriasis orinflammatory bowel disease. Conditions in which immune suppression wouldbe advantageous include conditions in which a normal or an activatedimmune response is disadvantageous to the mammal, e.g.allo-transplantation of e.g. body fluids or parts, to avoid rejection,or in fertility treatments in which inappropriate immune responses havebeen implicated in failure to conceive and miscarriage. The use of suchcells before, during, or after transplantation avoids extensive chronicgraft versus host disease which may occur in patients being treated(e.g. cancer patients). The cells may be expanded immediately afterharvest or stored (e.g. by freezing) prior to expansion or afterexpansion and prior to their therapeutic use. The therapies may beconducted in conjunction with known immunosuppressive therapies.

As used herein, “treating” refers to the reduction, alleviation orelimination, preferably to normal levels, of one or more of the symptomsof the disease or condition which is being treated, e.g. alleviation ofimmune dysfunction or avoidance of transplant rejection, relative to thesymptoms prior to treatment.

Preferably the mammal is a domestic or livestock animal (e.g. cats,dogs, rabbits or horses, pigs, cows, goats, sheep) or a primate, e.g. ahuman.

Alternatively viewed, this aspect of the invention also provides the useof a T cell population obtained by the stimulation method of the presentinvention in the preparation of a therapeutic composition for achievingan immunosuppressive effect in a mammal, e.g. for treating a mammal witha condition or disease typified by an aberrant immune response or inwhich immune suppression would be advantageous.

Alternatively, aspects of the invention provide the use of a TCR/CD3activator, a TCR co-stimulatory activator, and/or rapamycin in thepreparation of a medicament comprising a T cell population obtained bythe stimulation method of the present invention for achieving animmunosuppressive effect in a mammal, e.g. for treating a mammal with acondition or disease typified by an aberrant immune response or in whichimmune suppression would be advantageous.

Methods of obtaining a T cell sample from a patient (or an animal sourceif appropriate) and isolating CD4+ cells for expansion/activation arewell known and documented in the art. For example, peripheral bloodmononuclear cells can be obtained from buffy coats of normal blooddonors by density gradient centrifugation and leukopheresis. Adherentmononuclear cells can then be separated from non-adherent mononuclearcells by adherence to a solid surface such as for example beads ortissue culture plastic under appropriate conditions (e.g. 30 minutes to2 hours at 37° C.). Enriched T cells can be used directly for subsequentprocedures or T cells can then be separated from other non adherentmononuclear cells by a technique known as “rosetting”. Negativeselection of cells can also be used. Isolation of sub-populations of Tcells can then be carried out with the aid of appropriate antibodies tocell surface antigens displayed on the surface of particular populationsof T cells. Such techniques are sometimes referred to as positive ornegative selection.

Once an appropriate T cell population or sub population has beenisolated from a patient or animal, genetic or any other appropriatemodification or manipulation may optionally be carried out before theresulting T cell population is expanded using the methods and supportsof the invention. The manipulation may for example take the form ofre-stimulation of the T cells with anti-CD3 and anti-CD28 antibodies tore-activate them.

Genetic or other manipulation of the T cells is an optional step and isnot necessary for some therapies.

A yet further aspect of the invention provides a kit forexpanding/activating CD4+CD25− T cells comprising a T cell receptor(TCR)/CD3 activator, a TCR co-stimulator activator and rapamycin.Preferably the activators are CD28 antibodies and CD3/TCR antibodies,which are preferably immobilized on a solid support.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor(s) to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the scope of theinvention.

EXAMPLES Example 1 General Expansion Protocol for Generation ofCD4+CD25+FOXP3− Regulatory T Cells

1) Isolate CD4+ T cells from peripheral blood or leukopheresis productsby the use of Dynal® CD4 Positive isolation kit, Dynal® CD4 NegativeIsolation kit, Dynabeads® MyPure™ CD4 T cell kit, or CD4 MicrobeadsMACS®.

2) Stimulate 1.5×10⁶ CD4+ T cells with anti-CD3/CD28 Dynabeads (3:1bead:T cell ratio) in X-vivo medium with 1000 U/ml IL-4 (optional), 20U/ml IL-2 and 1 μM final concentration of Rapamycin. Total medium volumeshould be 1.5 ml per 1.5×10⁶ CD4+ T cells in a 24 well plate. Theprotocol can be scaled up.

3) On day 2, add a 10× concentrate that contains 10,000 U/ml IL-4(optional) and 10,000 U/ml IL-2. Add this in a volume that is 10% of theinitial culture volume.

4) On day 4, add the cytokine concentrate similar to day 2.

5) On day 6, add the 10× cytokine concentrate again and take an aliquotto count the cells. This is the first time that the culture should bedisturbed. Based on the count adjust the cell concentration to 1×10⁶ CD4cell/ml. Split wells and add culture medium containing Rapamycin, 1000U/ml IL-4 (optional), 20 U/ml IL-2 to maintain cell concentration at0.5×10⁶ cells/ml.

6) From day 7 to day 12 count the cells daily. Split wells and addculture medium containing Rapamycin, 1000 U/ml IL-4 (optional), 20 U/mlIL-2 to maintain the cell concentration at 0.5×10⁶ cells/ml. On day 12the beads are removed using a magnet.

7) On day 14-15 the cells can be analyzed for surface marker expressionand functional properties.

Example 2 Analysis of Properties of Expanded T Cells

CD4+ T cells were isolated from peripheral blood by negative and/orpositive isolation as described in Example 1. After expansion withIL-2,+/− IL-4, CD3/CD28 Dynabeads®, with or without Rapamycin theexpanded cells were analyzed for various surface markers (CD4, CD25,CD62L, CRTh2, CCR4, CXCR3, CCR7) and for intracellular FOXP3 expressionand cytokine secretion. Suppressive capacity was analyzed in standardfunctional assays as described below.

The above mentioned surface markers are as follows:

CD62L: Lymph node homing adhesion molecule, expressed on naive T cellsand naturally occurring CD4+CD25+ Treg cells.

CRTh2: A marker expressed on all Th2 cells.

CCR4: Chemokine receptor expressed on Treg cells and Th2 cells.

CXCR3: Chemokine receptor expressed on inflammatory Th1 cells.

CCR7:Lymph node homing chemokine receptor expressed on naïve T cells,central memory T cells and naturally occurring CD4+CD25+ Treg cells.)

CD4+ T cells expanded without Rapamycin show low expression of CD25,CD62L and CCR7, and higher expression of CXCR3. The cytokine profileshows high levels of both Th1 and Th2 cytokines. In contrast, the CD4+ Tcells expanded with Rapamycin have a phenotype of Treg cells with highCD25, low CXCR3 and low secretion of cytokines. Approximately 70% of theexpanded cells express high levels of CD25. None of the expanded T cellswith or without Rapamycin have an increased expression of FOXP3. 10% offreshly isolated CD4+ T cells expressed FOXP3 and after expansion stillonly 10% of the cells expressed FOXP3.

Standard proliferation assays were performed using 75,000 CD25− T cellssupplemented with CD25+ T cells to provide the ratios indicated in FIG.1 and thymidine incorporation was analyzed after a period of 4 days. Tcells expanded with Rapamycin (CD25+) were found to be capable ofsuppressing proliferation of non Rapamycin expanded T cells (CD25−). Inthe presence of CD25+ T cells, the CD25− T cells capacity to proliferatewas inhibited by 90%. (FIG. 1).

FIG. 2 illustrates the expression of CD4 and CD25 in CD4+ T cellsexpanded with or without rapamycin. Rapamycin expanded cells express 80%CD4+CD25+ T cells wherein in the absence of rapamycin only 20% of the Tcells were CD4+CD25+.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from the conceptand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the scope and concept of the invention.

1. A method of generating immunosuppressive regulatory T cells from asample, the method comprising: providing an initial sample containing apopulation of CD4+CD25− T cells; and contacting the population with a Tcell receptor (TCR)/CD3 activator, a TCR co-stimulator activator, andrapamycin to generate a final sample comprising immunosuppressiveregulatory T cells.
 2. The method of claim 1, further comprisingpurifying the population prior to the contacting step.
 3. The method ofclaim 2, wherein the purifying step comprises isolating CD4+ T cells. 4.The method of claim 1, wherein the generated immunosuppressiveregulatory T cells are CD4+CD25+.
 5. The method of claim 1, wherein thegenerated immunosuppressive regulatory T cells are CD4+CD25+FOXP3−. 6.The method of claim 1, wherein the population of CD4+CD25− T cellscomprise CD4+CD25−CD8+ T cells.
 7. The method of claim 1, wherein thesample is blood.
 8. (canceled)
 9. (canceled)
 10. The method of claim 1,wherein the population is contacted with the rapamycin before the T cellreceptor (TCR)/CD3 activator and TCR co-stimulator activator. 11.(canceled)
 12. The method of claim 1, wherein the rapamycin is added tothe population at a concentration of about 0.01 μM to about 10 μM. 13.(canceled)
 14. (canceled)
 15. The method of claim 1, wherein thecontacting step further comprises contacting the population with atleast one cytokine or growth factor.
 16. (canceled)
 17. The method ofclaim 1, wherein the T cell receptor (TCR)/CD3 activator is an antibodyor a ligand for TCR/CD3.
 18. (canceled)
 19. (canceled)
 20. The method ofclaim 1, wherein the TCR co-stimulator activator is an antibody. 21.(canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. The methodof claim 1, wherein: the T cell receptor (TCR)/CD3 activator isimmobilized on a solid phase; and the TCR co-stimulator activator isimmobilized on a solid phase.
 26. The method of claim 25, wherein the Tcell receptor (TCR)/CD3 activator and the TCR co-stimulator activatorare immobilized on the same solid phase.
 27. The method of claim 25,wherein the T cell receptor (TCR)/CD3 activator and the TCRco-stimulator activator are immobilized on different solid phases. 28.The method of claim 1, wherein: the T cell receptor (TCR)/CD3 activatoris immobilized on beads; and the TCR co-stimulator activator isimmobilized on beads.
 29. (canceled)
 30. (canceled)
 31. (canceled) 32.(canceled)
 33. The method of claim 25, wherein the solid phase comprisesspherical beads having diameters of about 1 μm to about 10 μm. 34.(canceled)
 35. (canceled)
 36. (canceled)
 37. A kit for the generation ofimmunosuppressive regulatory T cells, the kit comprising: a T cellreceptor (TCR)/CD3 activator, a TCR co-stimulator activator, andrapamycin.
 38. The kit of claim 37, wherein: the T cell receptor(TCR)/CD3 activator is immobilized on a solid phase; and the TCRco-stimulator activator is immobilized on a solid phase.
 39. A method oftreating a mammal, the method comprising: providing a first mammal;obtaining CD4+CD25− T cells from the first mammal; expanding/activatingthe T cells ex vivo by contacting the T cells with a T cell receptor(TCR)/CD3 activator, a TCR co-stimulator activator, and rapamycin; andadministering the expanded/activated T cells to a second mammal to betreated.